Systems for Expression of Heterologous Proteins in M. Capsulatus

ABSTRACT

The present invention relates to an expression system for the expression of proteins and peptides in a methanotrophic bacterium, preferably  M. capsulatus . Further, the invention relates to the exportation and display of said peptides and proteins on the surface of said bacteria. The invention also describes a method for the production of a desired protein in  M. capsulatus.

The present invention relates to the expression of heterologous proteins in the bacteria M. capsulatus. More specifically, the present invention relates to the exportation and display of polypeptides and proteins on the surface of said bacteria.

Thus, the present application relates to a recombinant vector, a bacterial host cell transformed with said vector, a method for producing a desired protein in a bacterial host cell, a protein capable of being exposed on the surface of a methanotrophic bacterium, and a fusion protein.

The expression of polypeptides on the surface of bacteria and bacteriophages has been pursued for several years, in part because of interest in recombinant antibody production. Many other potential applications exist, including the production of genetically-engineered whole cell adsorbents, construction of “peptide libraries”, cell bound enzymes, and use as live vaccines or immunogens to generate antibodies.

In bacteria, one approach to obtaining surface expressed foreign proteins has been the use of native membrane proteins as a carrier for a foreign protein. In general, most attempts to develop methods of anchoring proteins on a bacterial surface have focused on fusion of the desired recombinant polypeptide to a native protein that is normally exposed on the cell's exterior with the hope that the resulting hybrid will also be localized on the surface.

In a prior invention (Norwegian patent application no. 20033176) the present inventors also provided an expression system where a heterologous polypeptide (termed “desired” protein) was expressed in the bacteria Methylococcus capsulatus. The heterologous protein is preferably linked to an outer membrane protein in M. capsulatus termed MopE.

MopE has a 540 amino acid protein sequence, with a short (29 amino acids) N-terminal sequence dependent signal sequence, followed by a N-terminal domain (176 amino acids) and a C-terminal domain (335 amino acids). The N-terminal domain is not secreted, while the C-terminal domain is secreted and expressed on the cell surface. The MopE amino acid sequence is shown in the sequence listing, as Seq. ID. no. 1

The method of secretion is not known. MopE does not show high sequence similarity to other known secreted proteins. The secretion is host specific, although inserted in the IM and released to the periplasm, the MopE protein is not secreted by E. coli hosts, only by M. capsulatus. MopE has been proposed to be secreted either by the Type 2 Secretion system (T2S) or the Type 5 Secretion system (T5S) based upon its primary sequence (Fjellbirkeland et al., 2001). Proteins secreted by the T2S or T5S systems can be translocated across the IM either by the Tat or the Sec machinery, which are shown in FIG. 1.

Secretion of T2S substrates is very host specific. Secretion by the T2S system (FIG. 1) is a two-step process in which the secreted protein initially is exported across the IM by the Sec or Tat export systems. The periplasmatic intermediate is subsequently translocated across the OM by the T2S secretion through the channel formed by the secretin that is large enough to translocate folded or close to folded substrates. T2S is energized by ATP, and although not required for secretion, the proton motive force increases the rate of secretion. Substrates of the T2S system share no obvious similarities in primary sequence. Although no recognition signal that confine proteins to secretion by the T2S pathway has been identified, a potential common feature for T2S substrates is a medium to high content of β-sheet (de Vries et al., 1990 and Sandkvist, 2001). β-strands were predicted in the N-terminal domain of MopE by the computer program PRED-TMBB (http://bioinfromatics.biol.uoa.gr/PRED-TMBB), and this program also predicted an N-terminal β-barrel in MopE. The predicted β-strands may form the tertiary structures of β-sheets required for translocation by the T2S pathway. In addition to the prediction of such structures in N-terminal domain of Mop-E, MopE^(C) is heat-modifiable while MopE* is not (Fjellbirkeland et al., 2001), indicating that the N-terminal domain of MopE indeed contains stabile β-structures, and thus is a candidate T2S substrate. Since a Sec-compatible signal sequence has been predicted in MopE it has been considered likely that the Sec machinery export MopE across the IM.

However, the presence of β-structures does not confine secreted proteins to the T2S route. Substrates of T5S, autotransporters, require a β-domain that forms a β-barrel that allows for translocation of the passenger domain across the OM. Since the N-terminal domain of MopE has the potential of containing β-structures, the domain could function as an autotransporter translocation unit in T5S, No autoproteolytic activity could be demonstrated for MopE using the substrate azocasein, however, such activity is not a widely distributed feature among T5S substrates, and thus not required. And while autotransporters inherently contain all information and accessory factors necessary for translocation to the OM, release of the protein to the extracellular space most often require cleavage by an external protease. Thus secretion by T5S is somewhat dependent on the host cell, and the host specificity observed for the secretion of MopE does not exclude secretion by the T5S route.

Thus, it was thought that the most likely mechanism for translocation of MopE was either by the T2S or T5S routes, by the Sec or Tat transport systems. This translocation would then require the β-structures found in the N-terminal domain of MopE. Thus it was expected that the removal of the N-terminal domain of MopE would abolish the ability of the truncated protein, here termed MopE^(H)*, to translocate. The MopE^(H)* amino acid sequence is shown in the sequence listing, as Seq. ID. no. 2

The inventors constructed a mutated MopE protein, MopE^(H)*, that consisted of the secreted domain of MopE alone, and did not contain the N-terminal MopE domain. (Please see FIG. 2. for a comparison of MopE and MopE^(H)*) Quite surprisingly, they found that MopE^(H)* fully retained its' ability to translocate in M. capsulatus.

Experiments were also performed to acertain the usefulness of MopE^(H)* as part of a fusion protein capable of translocation, by expressing Atlantic halibut nodavirus capsid protein fused to the surface protein MopE. As shown below, these fusion proteins were indeed capable of translocation.

As stated, this translocation of MopE^(H)*, with or without a fused protein, is very surprising because it was thought that the N-terminal of MopE was required for translocation by the T2S or T5S routes. In addition, even though a protein in its entirety, here MopE, is known to be translocated, there is no reason to believe that only parts of the protein also may be able to do so.

The importance of this invention is fairly self evident. If one is to use MopE as a translocation system, there is an advantage to narrow it down to the smallest possible protein. There are always limits to how large of a protein (i.e. how long of a amino acid chain) may be transported. By removing a part of the native protein (here the N-terminal domain) one may, as shown, fuse a proportionally larger protein to the truncated MopE protein, and still have a reasonable expectation of successful translocation. In addition, the discovery that the N-terminal is not involved in translocation, changes the understanding of how MopE is translocated, and points research in new directions in trying to ascertain the mechanisms behind the translocation.

The invention can be better understood with reference to the following figures:

FIG. 1. Overview of secretion by the Type 2 and Type 5 secretion systems. (A) The T2S system secretion consists of 12-16 proteins depending of species. The majority of the T2S components are IM proteins, many with large periplasmatic domains. Two of these GspE and GspL interact with ATP and are involved in energizing of the secretion. In the OM 12-14 GspD and GspS units form a secretin. The secretin is a transmembrane complex with a channel 5-10 nm in diameter, thus large enough to translocate folded or close to folded polypeptides. (B) Proteins secreted by T5S are pre-pro-proteins consisting of three domains: an N-terminal signal sequence for export across the IM, an internal passenger/functional domain and a C-terminal β-domain. The figures (A) and (B) are reproduced from (Voulhoux et al., 2001) and (Desvaux et al., 2004), respectively.

FIG. 2. Overview of the structure of MopE and Mope*.

MopE consists of two domains, the non-secreted N-terminal domain and the secreted C-terminal domain, in addition to a Sec-dependent signal sequence. In MopE^(H*) the histidine originating from the cloning strategy is shown.

FIG. 3. Amplification of DNA molecules containing the mopE^(H)* gene or the mmoX promoter linked to the sequence encoding the MopE signal sequence from pAFpg10 or pJBrp2, respectively. The mopE^(H)* gene was constructed by amplification of a region of pAFpg10 using the primers MopEXhoR and MopE*NcoI. The DNA containing the copper sensitive mmoX promoter and the sequence encoding the MopE signal sequence (MopE ss) was amplified from pJBrp2 using the primers sMMOSacI and spNcoI.

FIG. 4. Amplification DNAs containing the mopE^(H)* gene or the mmoX promoter linked to the sequence encoding the MopE signal sequence. Negative PCR controls used had composition identical to the PCR sample except that no template was added. (A) PCR amplification of the ˜0.5 kb DNA containing the mmoX promoter linked to the sequence encoding the MopE signal sequence (lane 2) and PCR negative control (lane 1). (B) PCR amplification of a ˜1.2 kb DNA containing the mopE^(H)* gene (lane 1) and PCR negative control (lane 2). (C) The amplified DNAs were subcloned in pCR2.1-TOPO vectors, and resulting plasmids were controlled by RE-analysis. NcoI/XhoI digested pCR2.1-TOPO1 (lane 1) and pCR2.1-TOPO2 (lane 2).

FIG. 5. Subcloning of the amplification products in pCR®2.1-TOPO vectors by TOPO® TA cloning. The DNA containing the mmoX promoter linked to the sequence encoding the MopE signal sequence was inserted to pCR2.1-TOPO to produce pCR2.1-TOPO1, while the DNA containing the mopE^(H)* gene was inserted to pCR2.1-TOPO to produce pCR2.1-TOPO₂.

FIG. 6. Subcloning of the DNA containing the mmoX promoter linked to the sequence encoding the MopE signal sequence in pET11d. The DNA containing the mmoX promoter and the sequence encoding the MopE signal sequence was inserted to pET11d, producing pET1.

FIG. 7. Construction of and RE-analysis of pET1 (A) The ˜0.5 kb DNA containing the mmoX promoter linked to the sequence encoding the MopE signal sequence (lane 1) and the ˜5.9 kb pET11d vector fragment (lane 2) were purified from an agarose gel. (B) pET1 was digested by BamHI (lane 1) and double digested with NcoI and XbaI (lane 2).

FIG. 8. Subcloning of the DNA containing the mopE^(H)* gene in pET1. To produce pET2 a DNA containing the mopE^(H)* gene was inserted to pET1.

FIG. 9. Construction of and RE-analysis of pET2. (A) A ˜1.2 kb DNA containing the mopE^(H)* gene (lane 2) and a ˜6.3 kb pET1 fragment (lane 1) were purified from an agarose gel. (B) The pET2 plasmid was digested by BamHI (lane 1) and double digested by BamHI and NcoI (lane 2).

FIG. 10. Construction of pBBmopE^(H)*. A pET2-DNA containing the mopE^(H)* gene, the mmoX promoter and the sequence encoding the MopE signal sequence was excised from pET2 an inserted to pBBR1MCS-2 to produce pBBmopE^(H)*.

FIG. 11. Construction and RE-analysis of pBBmopE^(H)*. (A) The ˜5.1 kb pBBR1MCS-2 fragment (lane 1) and ˜2.0 kb pET2 (lane 2) were purified from a preparative agarose gel. (B) The pBBmopE^(H)*plasmid was double digested with BamHI and XbaI (lane 1) and single digested by NdeI (lane 2).

FIG. 12. ECL developed blot of spent media of a copper-depleted M. capsulatus (Bath) wild type culture (lane 1) and of copper-depleted cultures of M. capsulatus ΔmopE harbouring either pBBR1MCS-2 (lane 2) or pBBmopE^(H)*(lane 3).

FIG. 13. Diagrams of MopEH*, and MopEH* modified to contain restriction sites for BspHI and NheI, in order to facilitate cloning of fusion proteins.

FIG. 14. The three MopEH*-nodavirus constructs developed: AHNVC-MopEH*, MopEH*-AHNVCc and MopEH*-AHNVC-20.

FIG. 15. Schematic of the determination of the localization of recombinant MopEH*

FIG. 16. Gel results of MopEH*-AHNVCc (16 a) and MopEH*-AHNVC-20 (16 b) and AHNVC-MopEH* (16 c) using anti-AHNVC and anti-MopEH*.

FIG. 17. Use of antibodies against MopEH*-AHNVC 20 aa peptide shows that the construct is antigenic.

Cultured Methylococcus capsulatus with pBBmopE^(H)* plasmid was deposited with DDZ access identification Methylococcus capsulatus McdeltamopEpBBmopEH=DSM 19108 on Mar. 1, 2007 with DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (The German National Resource Centre for Biological Material), Inhoffenstr. 7 B, D-38124 Braunschweig, Germany.

A sequence listing was prepared using Patentln3.4 for the amino acid sequences of MopE (Seq. ID no. 1) and MopEH* (Seq. ID no. 2), AHNVC-MopEH* fusion protein (Seq. ID no 11), MopEH*-AHNVCc fusion protein (Seq. ID no 13), MopEH*-AHNVC-20 aa fusion protein (Seq. ID no 15), and the DNA sequences of MopE (Seq. ID no. 3), MopEH* (Seq. ID no. 4), pET1 (Seq. ID no. 5), pET2 (Seq. ID no. 6), pCR2.1-TOPO1 (Seq. ID no. 7), pCR2.1-TOPO2 (Seq. ID no. 8), and pBBmopEH* (Seq. ID no. 9), AHNVC-MopEH* (Seq. ID no 10), MopEH*-AHNVCc (Seq. ID no 12), MopEH*-AHNVC-20 aa (Seq. ID no 14), MopEH* with BspHI mutation in stop codon (Seq. ID no 16), MopEH* with NdeI mutation (Seq. ID no 17), Atlantic halibut Nodavirus capsid protein 2 (Seq. ID no 18) and pBBR1-mopEH*(Seq. ID no 19). The primers given in tables 4 and 6 are also given as PatentIn sequences ID no 20-33. MopEH ST25.txt PatentIn utskrift

Thus, the recombinant vector according to the present invention comprise a first nucleotide sequence Seq. ID no. 4, or sequences homologous thereto, capable of translocation through the outer membrane of Methylococcus capsulatus.

Also, the bacterial host cell according to the present invention is transformed with said recombinant vector.

And, the method for producing a desired protein in a bacterial host cell according to the present invention comprise transforming a bacterial host cell with a recombinant vector comprising a first nucleotide sequence Seq. ID no. 4 or sequences homologous thereto, and said vector comprising a further nucleotide sequence encoding said protein, said further nucleotide sequence being operably linked in frame to said first nucleotide sequence, and culturing said transformed host cell in a suitable medium under conditions allowing expression of said protein.

And, the protein capable of being exposed on the surface of a methanotrophic bacterium according to the present invention is encoded for by Seq. ID no. 2 or sequences homologous thereto.

And, the fusion protein according to the present invention comprise a protein or peptide sequence encoded for by a nucleotide sequence Seq. ID no. 4 or sequences homologous thereto, and a desired protein or peptide, capable of being translocated.

The fusion protein according to the invention is preferably expressed from a chimeric DNA having a DNA segment encoding a leader amino acid sequence capable of mediating secretion of the fusion protein, a DNA segment encoding for subunits of the surface protein, and a DNA segment encoding the desired target protein. The DNA segments are positioned such that expression of the fusion protein results in display of the target protein on the surface of the cells. The fusion proteins are preferably anchored to the cell surface of the bacteria forming what is referred to as a “display bacteria.”

The present invention thus provides for a system for the expression of heterologous proteins, where the heterologous proteins are expressed on the surface of the bacterial cells.

The chimeric DNA may be integrated into the bacterial cell chromosome or be carried by a vector, where said vector preferably dose not comprise the entire nucleotide sequense of MopE (Seq. ID no 3), but rather only the truncated sequence comprising MopE^(H)*(Seq. ID no 4). In certain preferred embodiments, expression of the fusion protein may be regulated by an inducible promoter. Bacteria displaying a particular protein may be selected, for example, using antibody affinity. The fusion protein can be detached from selected cells. If desired, the target protein may be separated from the surface protein and further purified.

Target proteins useful in the present invention include peptides, proteins, e.g., hormones, enzymes, inhibitors, and receptors, antigens, antibodies including antibody fragments and single-chain antibodies.

The present invention thus provides a system for the expression of heterologous proteins in the membrane fraction, and preferable on the cell surface of the M. capsulatus.

The bacterium M. capsulatus is able to utilise methane as a single carbon and energy source. Bacteria capable of oxidising methane are collectively referred to as methanotrophs. They belong to different families and groups of the eubacteria but have in common the possession of the unusual enzyme methane mono oxygenase, which catalyses the oxidation of methane to methanol.

The bacterium has an obligate requirement for methane or methanol and an optimum growth temperature of 45° C. Methane is oxidized via methanol to formaldehyde which is either assimilated into cellular biomass or dissimilated to carbon dioxide to release cellular energy.

M. capsulatus has a gram-negative cell envelope. Much of the intracellular space is occupied by an extensive intracytoplasmic membrane system. The genome of M. capsulatus (Bath) has a molecular weight of 2.8×10⁹ Da and a G+C content of 62.5%.

Commercial interests involving M. capsulatus and other methanotrophs could roughly be divided into two categories: Those taking advantage of the inexpensive growth requirements of the bacteria and those taking advantage of unique catalytic activities possessed by the bacteria.

The development of high-cell density fermentation technology for M. capsulatus has created the possibility of producing large quantities of specialised compounds like for instance amino acids, cofactors, vitamins, metabolic end products, and various high value proteins, at reasonable costs.

The present invention thus provides a system for the manufacturing of such product.

Other uses for the protein display methods of the present invention include, for example, epitope mapping, screening of antibody libraries and live bacterial vaccines.

The invention is especially suited for production of vaccines that can be administered orally for use in animals, fish and humans. The technique can also potentially be used for display of vaccines, especially for oral administration.

The invention relates to the use of the genes and the proteins encoded by them, as given in the accompanying sequences list, fragments thereof, or functionally equivalent substantially similar genes, for construction of fusion proteins carrying foreign peptide sequences for display in the M. capsulatus, and preferable on the surface of said bacterium. The term “homology” or “homologous”, as used in the present application, does not necessary infer a common evolutionary ancestor/relationship, as homologous sequences may be artificially created. Rather, it is meant to encompass sequences that are similar and have a similar function, that is, sequences likely to be able to perform the same/similar function due to having a degree of sequence similarity (that may be defined as a percentage sequence similarity/identity).

M. capsulatus is a bacterium licensed for use in animal and fish feed. It has no virulent or pathogenic properties, and contains very low amounts of endotoxin (LPS). It is thus well suited as a carrier organism for recombinant oral vaccines, with a potential also for use in humans. Vaccines could be constructed by insertion of fragments of D15 genes from pathogens into the M. capsulatus D15 gene in order to display a fusion-protein containing parts of the two D15 antigens on the surface of M. capsulatus. The part of the D15 protein originating from the pathogen should trigger an immune response to the respective pathogenic bacterium. If replacement of M. capsulatus-specific D15 sequences with corresponding sequences from the pathogens is well tolerated by the host, larger regions of D15 could be replaced, and if possible, the entire D15 protein could be replaced by the corresponding protein from a pathogen.

Due to the sequence conservation of D15 among distantly related bacteria, exchange of parts of the gene (or the entire gene) without seriously affecting the survival and growth of M. capsulatus is plausible. The specific function of the D15 antigen on the surface of the bacteria is not known, but it possibly plays a structural role and is most probably not involved in any important biochemical processes.

Successful display of the target protein on the cell surface can be detected using a number of methods, for example, if the target peptide can be specifically labeled by a procedure that does not operate through the membrane, its cell surface display can be readily demonstrated.

If the target polypeptide displays enzymatic activity, one may use such activity to demonstrate cell surface display. Antibodies against the target protein may also be used.

The chimeric DNA may be integrated into the host cell chromosome or be carried within a vector. Methods of integrating DNA into a host cell chromosome are well known in the art. The chimeric DNA may also be carried within a recombinant vector, e.g., a plasmid.

Plasmids useful as the vector backbone include plasmids containing replicon and control sequences which are derived from species compatible with the host cell. The vector may also contain an inducible promoter and marker gene, e.g., antibiotic resistance.

Introduction of the chimeric DNA to the host cell may be effected by any method known to those skilled in the art. For example, if a recombinant vector carries the DNA, the vector can be introduced, for example, by transformation, electroporation, or phage transfection.

The detection techniques noted above can be used initially to verify that the method of the present invention is working, i.e., that the fusion surface protein has been expressed and transported to the bacterial cell surface and is orientated so that the target protein is accessible i.e., displayed.

Cells that display the target may be separated from those that do not, using, for example, affinity separation techniques. Such techniques include affinity column chromatography, batch elution from affinity matrix material and fluorescent-activated cell sorting.

MopE is a major outer membrane protein of M. capsulatus. It contains surface-exposed regions but its exact folding and association with the cell surface is not known. Under copper limitations, the C-terminal part of the protein is secreted into the growth medium, but considerable amounts of the full-length protein remains associated with the cell surface. By using this protein as an anchor it is possible to mediate translocation of passenger proteins to the cell surface or to the extracellular environment.

EXPERIMENTAL SECTION Bacterial Strains M. Capsulatus

TABLE 1 Strains of M. capsulatus used. Strain Description and use Reference M. capsulatus (Bath) wild Used as control and reference. Whittenbury et type NCIMB 11132 al., 1970 M. capsulatus ΔmopE Contains an inactivated mopE gene and is Fjellbirkelan gentamycin resistant. Used as host for unpublished plasmids expressing mutated MopE protein and as mating-pair recipient in conjugation with E. coli S17-1

E. Coli

TABLE 2 Stains of E. coli used. Strain Description and use Reference One Shot Genotype: F′ [lacI^(q)Tn10 (Tet^(R))] mcrA ?(mrr- hsdRMS- Invitrogen TOP10F′ mcrBC) Φ80lacZ?M15 ?lacX74 recA1araD139 ?(ara- leu)7697 galU galK rpsL (Str^(R)) endA1 nupG Used as intermediate host for constructed plasmids, and as host for TOPO TA cloning. S17-1 Genotype: Tp^(R) Sm^(R) recA, thi, pro, hsdR-M + RP4: 2-Tc:Mu: Simon et al., Km Tn7 1 pir 1983 Has genomically inserted tra genes and were used as mating-pair donor in conjugation with M. capsulatus ΔmopE. DH5α Genotype: supE44 DlacU169 (F80 lacZDM15) hsdR17 Invitrogen recA1 endA1 gyrA96 thi-1 relA1 Host for plasmids pAFpg10 and pJBrp2.

Plasmids

TABLE 3 List of plasmids used. Name Description and use Reference pAFpg10 Contained both mopE and Amp^(R). Used as template for PCR Fjellbirkeland et amplification of the truncated mopE gene, mopE^(H)*, and for site-directed al., 2001 mutagenesis to create mopE genes mutated by substitution, the mopE^(dn) genes. pJBrp2 Contained Km^(R), the copper-sensitive mmoX promoter and the sequence Haugland, encoding the MopE signal sequence. unpublished Used for PCR amplification of the mmoX promoter and the sequence encoding the MopE signal sequence. pCR ®2.1 Used in subcloning of an amplification product containing mmoX INVITROGEN promoter and the sequence encoding the MopE signal sequence or mopE^(H)* by TA TOPO cloning. Linear and with 3′-T overhang. Contained lacZα, Km^(R), Amp^(R), F1 ori and pUCori. pCR ®2.1 TOPO1 Cloning intermediate based on pCR ®2.1 that contain the mmoX promoter This application linked to the sequence encoding the MopE signal sequence. Also contained Km^(R), Amp^(R) and LacZα disrupted by insertion of the DNA fragment. pCR ®2.1 TOPO2 Cloning intermediate based on pCR ®2.1 that contain mopE^(H)*. Also This application contained Km^(R), Amp^(R) and LacZα disrupted by insertion of the mopE^(H)* gene. pET11d Intermediate vector used to connect the mmoX promoter, the sequence Stratagene encoding the MopE signal sequence and the mopE^(H)* gene. Also contained Amp^(R), pBR322 ori, lacIq and lac operator. pET1 Cloning intermediate based on pET11d that contain the mmoX promoter This application linked to the sequence encoding the MopE signal sequence. Also contained Amp^(R). pET2 Cloning intermediate based on pET11d that contain the mmoX promoter This application linked to the sequence encoding the MopE signal sequence and the mopE^(H)* gene. Also contained Amp^(R). pBBR1MCS-2 Used as vector for the mopE^(H)* gene. Contained mob genes, thus were Kovach et al., mobilizable when tra genes were provided by E. coli S17-1. Also 1995 contained lacZα, Km^(R) and rep. pBBmopE^(H)* Used to express mopE^(H)* in E. coli S17-1 and to transfer mopE^(H)* to M. This application capsulatus ΔmopE. Vector based on pBBR1MCS-2 containing a truncated mopE, mopE^(H)*, the mmoX promoter and the sequence encoding the MopE signal sequence. Also contained Km^(R).

Primers

TABLE 4 List of primers used. Name Sequence Use sMMOprSacI 5′-GTGGAGCCGTTGCCGTTC PCR amplification of CGGTTCAGCGTGTCC-3′ mmoX promoter linked to the sequence encoding the MopE signal sequence MopEXhoR 5′-TGGCGGTGATCTCGAGCC PCR amplification of TGC-3′ mmoX promoter linked to the sequence encoding the MopE signal sequence spNcoI 5′-AGTGCCTCCATGGGCGGC PCR amplification of TG-3′ the mopE^(H)* gene. MopE*NcoI 5′-CAGCGAACTCCCATGGCC PCR amplification of TGGAC-3′ the mopE^(H)* gene.

Eurogentec supplied all primers, except from MopEXhoR supplied by TAGN Ltd and M13 forward supplied by Invitrogen.

Kits

TABLE 5 List of kits used. Kit Use Supplier QIAQuick Miniprep Purification of plasmid DNA QIAGEN QIAGEN HiSpeed Midi Plasmid Large scale purification of QIAGEN Purification Kit plasmid PCR purification Kit Purification of PCR products QIAGEN TOPO TA cloning Kit Subcloning of PCR products Invitrogen QIAQuick Gel Extraction KIt Extraction of DNA from QIAGEN agarose gels ECL western blotting detection system Development of immunoblot Amersham Bioscience Transfer of Plasmid DNA to M. capsulatus by Conjugation

Presently conjugation is the only method available for transfer of genetic information to Methylococcus. Conjugative transfer require establishment of physical contact between the cells of the mating-pair, the DNA donor and the DNA recipient. Additionally, the donated plasmids must hold mob or tra genes. The plasmid used, pBBR1MCS-2 (Table 3-3), contained mob genes, while E. coli S17-1 contained tra genes.

Conjugation was performed as described by Lloyd et al (1999) using the plasmids derived from the mobilizable plasmid pBBR1MCS-2 and the mating-pair donor E. coli S17-1.

M. capsulatus and E. coli whole cells were then separated from the spent medium by centrifugation.

Concentration of Spent Medium Proteins by Cellulose Ultrafiltration

MopE* is the major protein detectable in unconcentrated spent medium of M. capsulatus cultures. Spent medium proteins were concentrated by cellulose ultrafiltration using the Amicon® Ultra-15 PL-100 centrifugation filter device. The filter used had a nominal molecular weight limit of 10 kDa and maximum sample volume of 15 ml. Spent medium from a 150 ml cultures was concentrated to a final volume of about 200 μl by repeated centrifugations.

Strategy for Cloning

Genetic manipulation in M. capsulatus imposes several constraints regarding systems available for genetic transfer. Conjugation is the only method known to be effective in transferring genes to M. capsulatus, thus the conjugative vector, pBBR1MCS-2, was chosen as carrier of the mutated mopE genes. Based on its successful use in prior conjugations to M. capsulatus, E. coli S17-1 was chosen as plasmid DNA donor. When this study was initiated expression vectors compatible with M. capsulatus were not available. Thus, to enable initiation of transcription in M. capsulatus, a promoter recognizable by this bacterium was connected to the mutated mopE genes in the conjugative plasmids. Moreover, it was desirable that the transcription should be regulated in a relatively easy manner and this led to use of the mmoX promoter. The mmoX promoter initiate transcription of the M. capsulatus (Bath) operon mmoXYBZYC, and its activity is affected by the concentration of copper. The promoter region used was the 335 bp region located immediately upstream of the start-codon of mmoXYBZYC. This region has been shown to be sufficient for the copper-dependent activity of the promoter.

Construction of the mopE^(H)* Gene that Encodes the MopE^(H)* Mutant Protein

The mopE gene was contained in pAFpg10 (Table 3.), and this plasmid was purified from cells from an E. coli DH5a culture. The deletion mutant of the mopE gene was constructed by PCR amplification (FIG. 3.) using the forward primer MopE*NcoI (Table 4.) and the reverse primer MopEXhoR (Table 4.). The resulting amplified fragment (FIGS. 3. and 4.) was ˜1.2 kb and consisted of a DNA encoding MopE* (Gly₂₀₅-Pro₅₄₀), as well as a downstream region containing a Rho-independent transcription terminator. By using primers slightly non-complimentary to their target sequences, flanking NcoI and XhoI recognition sites were introduced in this amplified product. The introduction of the 5′ NcoI site resulted in addition of an additional histidine codon to the 5′-end of the mopE* gene. Thus, the amplified gene constructed and the protein encoded by it, was designated mopE^(H)* and MopE^(H)*, respectively.

The mmoX promoter was present in the plasmid pJBrp2 (Table 3.) linked to the sequence encoding the MopE signal sequence. This plasmid was purified from E. coli DH5α cells (Table 2.), and a fragment containing the mmoX promoter linked to the sequence encoding the MopE signal sequence was amplified from pJBrp2 by the PCR using the primers spNcoI (Table 4.) and sMMOprSacI (Table 4. and FIG. 3.). The resulting ˜0.5 kb DNA product (FIG. 4.) was flanked by Sad and NcoI restriction sites.

To simplify handling of the amplified fragments, the two amplification products were individually cloned into pCR®2.1-TOPO vectors (Table 3.) by TOPO® TA cloning producing pCR2.1-TOPO1 and pCR2.1-TOPO2 (FIG. 5.). Transformants from both the TOPO® TA reactions were selected based on their resistance to ampicillin and impaired production of β-galactosidase. A high yield of transformants was obtained from both transformation reactions.

A few single colonies of transformants were picked and cultivated in liquid LB. Cells from the E. coli TO10F′ cultures were harvested, plasmids were purified and analyzed by NcoI and Sad digestion (FIG. 4. C). All plasmids purified from the TOPO® TA cloning reaction with the ˜0.5 kb PCR product were digested into three fragments of lengths ˜0.5 kb, ˜1.5 kb and ˜2.5 kb, while all the plasmids purified from the TOPO® TA cloning reaction with the ˜1.2 kb PCR product were digested into three fragments of lengths ˜1.2 kb, ˜1.5 kb and ˜2.5 kb (FIG. 4. C lane 1-2, respectively). Thus, the results of the RE-analyses were in agreement with theoretical predictions. One E. coli TO10F′ colony containing pCR2.1-TOPO1 and one colony containing pCR2.1-TOPO2 were selected for further analysis. Plasmids from these colonies were purified and sequenced. This sequencing confirmed that the fragment containing the mmoX promoter linked to the MopE signal sequence fragment was contained in pCR2.1-TOPO1, and that the fragment containing the mopE^(H)* gene was contained in pCR®2.1-TOPO2.

Because of incompatibility of RE-sites in plasmid and fragments, the mopE^(H)* gene could not be connected to the mmoX promoter and the sequence encoding the MopE signal sequence directly in the conjugative vector pBBR1MCS-2. Thus, the fragments should be subcloned in pET11d (Table 4.). First, the DNA containing the mmoX promoter linked to the sequence encoding the MopE signal sequence should be inserted to pET11d (FIG. 6.) to produce pET1 (Table 3.). The DNA containing the mopE^(H)* gene then should be inserted to pET1 to produce pET2 (Table 3.). Thus, in pET2 the mmoX promoter should precede the mopE^(H)* gene connected with an upstream sequence encoding the MopE signal sequence (FIG. 2.).

As a first step to construct pET1, both pCR2.1-TOPO1 and pET11d were digested by NcoI and XbaI. The restriction of pCR2.1-TOPO1 produced three fragments with lengths ˜0.5 kb, ˜1.7 kb and 2.3 kb, while restriction of pET11d produced a ˜5.8 kb fragment, all in agreement with the theoretical predictions. The ˜0.5 kb DNA containing the mmoX promoter linked to the sequence encoding the MopE signal sequence and the ˜5.9 kb vector fragment were purified from the preparative agarose gel (FIG. 7. A lane 1-2), and used in a subsequent ligation reaction. The ligation solution was used to transform E. coli TOP10F′ cells and the resulting transformants were selected based on their resistance to ampicillin. One colony of transformed E. coli TOP10F′ cells was obtained. The transformed colony was cultivated in a 5 ml LB culture. A plasmid, designated pET1, was purified from the cells and analysed by RE digestion. The length of pET1 was estimated to be about 6.3 kb by agarose gel electrophoresis (FIG. 7. B lane 1). In agreement with theoretical predictions pET1 to produced two bands, one of length ˜5.8 kb and one of ˜0.5 kb after NcoI/XbaI double digestion (FIG. 7. B lane 2). Insertion of the mmoX promoter and the sequence encoding the MopE signal sequence were verified by sequencing.

To produce pET2 the DNA containing the mopE^(H)* gene was inserted to pET1 (FIG. 8.). The DNA containing the mopE^(H)* gene was excised from pCR®2.1-TOPO2 by digestion with BamHI and NcoI and this resulted in three fragments with apparent lengths of ˜1.2 kb, ˜1.6 kb and ˜2.3 kb, as theoretically predicted. The plasmid pET1 was opened by digestion with BamHI and NcoI and this resulted in a linear vector fragment of about 6.3 kb.

The ˜1.2 kb DNA containing the mopE^(H)* gene and the ˜6.3 kb pET1 fragment were purified from a preparative agarose gel (FIG. 9. A lane 1 and 2, respectively) and used in a subsequent ligation reaction. The ligation solution was used to transform E. coli TOP10F′ cells and transformants were selected based on resistance to ampicillin. A generous number of colonies of transformed E. coli TOP10F′ cells were obtained. A few colonies were picked and cultivated in liquid media for further analysis. Plasmids, designated pET2, were purified from the selected transformed cells and analysed by RE digestion. As predicted theoretically the length of the pET2 was estimated to be ˜7.4 kb by agarose gel electrophoresis (FIG. 9. B lane 1). Double digestion of the plasmid with BamHI and XbaI produced two DNAs, one ˜1.7 kb and one ˜5.7 kb fragment (FIG. 9. B lane 2), as theoretically predicted. Sequencing confirmed that the mopE^(H)* gene, in pET2, was preceded by the mmoX promoter and the sequence encoding the MopE signal sequence.

From pET2 a DNA fragment containing the mopE^(H)* gene proceeded by the sequence encoding the MopE signal sequence and the mmoX promoter could be excised and transferred to the mobilizable vector pBBR1MCS-2 (Table 3.). This would produce pBBmopE^(H)*(FIG. 10.). The pBBmopE^(H)* amino acid sequence is shown in the sequence listing, as Seq. ID. no. 9.

This pET2-DNA fragment was excised from the plasmid by restriction with HindIII and XbaI. As theoretically predicted this resulted in two DNAs, one of ˜2.0 kb and one of ˜5.5 kb. Also as expected, opening of pBBR1MCS-2 by digestion with HindIII and XbaI produced a ˜5.1 kb vector-DNA. The ˜5.1 kb vector-DNA was purified from a preparative agarose gel along with the ˜2.0 kb pET2-DNA containing the mmoX promoter linked to the sequence encoding the MopE signal sequence and the mopE^(H)* gene (FIG. 11. A lane 1 and 2) and were ligated. The ligation solution was subsequently used to transform E. coli Top10F′ cells, and transformed cells were selected based on their resistance to kanamycin. A total of 26 colonies of transformed E. coli Top10F′ cells were obtained. A few colonies of transformants were cultivated in 5 ml cultures for further analysis.

Plasmids, designated pBBmopE^(H)*, were purified from the selected transformed cells and analysed by RE digestion. As theoretically predicted the length of pBBmopE^(H)* was estimated to be about 7.2 kb by agarose gel electrophoresis (FIG. 11. B lane 2), while double digestion of pBBmopE^(H)* with BamHI and XbaI produced two DNAs, a ˜2.0 kb and a ˜5.1 kb DNA (FIG. 11. B lane 1). That pBBmopE^(H)* contained a mopE^(H)* gene preceded by the mmoX promoter and the sequence encoding the MopE signal sequence was confirmed by sequencing.

Production of MopE^(H)* in E. coli S17-1

A previous study in our laboratory has shown that the mmoX promoter is functional in E. coli. The expression of the mutated mopE^(H)*gene was studied in E. coli prior to transfer of the gene to M. capsulatus. E. coli whole cells and spent media were analysed by immunoblotting. E. coli S17-1 cells harbouring pBBmopE^(H)* were harvested from 50 ml cultures and By immunoblotting one immunoreactive protein migrating according to an apparent molecular mass of about 50 kDa was detected (not shown), thus the protein migrated shorter than wild type MopE* in the gel. Thus the E. coli host cell apparently produced MopE^(H)*, but the host was not able to cleave off the signal peptide. As expected no immunoreactive proteins were detected in the E. coli S17-1 cells harbouring pBBR1MCS-2 (not shown).

Spent medium from a culture of E. coli S17-1 harbouring pBBmopE^(H)* was concentrated. No immunoreactive proteins were detected in the concentrated spent medium. Thus, MopE^(H)* were seemingly not secreted from E. coli S17-1 in detectable amounts. As expected, the E. coli S17-1 pBB1MCS-2 did not either secrete immunoreactive proteins.

Production of MopE^(H)* in M. Capsulatus ΔmopE

The pBBmopE^(H)* plasmid was transferred to M. capsulatus:

The plasmid was transferred from E. coli S17-1 to M. capsulatus ΔmopE by conjugation and M. capsulatus cells transformed by pBBmopE^(H)* were selected by their resistance to kanamycin and gentamycin. A total of four conjugants were obtained. A few were selected for further analysis. The pBBmopE^(H)* plasmid was purified from the selected transformants and re-sequencing confirmed that no deletions had occurred during the conjugation process.

To study the expression of MopE^(H)* in M. capsulatus the copper-sensitive mmoX promoter was induced by cultivation of M. capsulatus ΔmopE containing pBBmopE^(H)* in a copper-depleted medium. Cells were separated from the spent medium by centrifugation, No MopE proteins could be detected in the M. capsulatus ΔmopE cells harbouring the empty conjugative plasmid, and no MopE protein was secreted from these cells.

The spent medium was isolated from the M. capsulatus ΔmopE pBBmopE^(H)* cell, concentrated and subjected to SDS-PAGE and immunoblotted. One immunoreactive protein was detected (FIG. 12. lane 3). This protein migrated as wild type MopE* (FIG. 12. lane 1), demonstrating that MopE^(H)* was secreted from M. capsulatus ΔmopE. Thus, the protein was properly processed in M. capsulatus and was able to cross the OM even though the N-terminal domain had been removed.

In conclusion, MopE^(H)* was expressed both in E. coli S17-1 and in M. capsulatus ΔmopE, but the secretion of MopE^(H)* was host specific, as MopE^(H)* was detected in the spent medium of the M. capsulatus culture only. This shows conclusively the abillety of MopE^(H)* to translocate across the outer membrane of M. capsulatus.

The inventors have in a previous application (Norwegian patent application no. 20033176) established a fusion protein of the complete MopE from M. capsulatus and the VP2 protein in of the infectious pancreatic necrosis (IPN) virus. They have also demonstrated that it is possible to express heterologous peptides in M. capsulatus by using the native protein MopE as a fusion partner. These fusion proteins did translocate, and produced immunological active antibodies.

Cloning of Nodavirus Capsid—MopEH* Fusion Proteins

In order to ascertain whether MopE^(H)* is able not only to translocate itself, but to do so as a functional fusion protein, several constructs with Nodavirus capsid protein were constructed, and the expression thereof was tested.

Atlantic halibut nodavirus is a RNA virus infecting mitochondria of insects or fish. It infect halibut at the larvae or juvenile stage, and mortality rates are up to 100%. Antibodies against AHNV have been previously developed.

The cloning of the fusion protein constructs were achived by conventional methods, including standard lab methods and commercial kits for cloning, mutagenese, immunoblotting etc, using pBBR1MCS2, as described above, as the starting plasmid. This time, instead of cloning in the MopE^(H)* nucleotide sequence alone, the sequence was first modified to comprise capsid protein from Atlantic halibut Nodavirus. In order for the capsid protein DNA sequence to be inserted into the MopEH* sequence, the MopEH* sequence was modified to comprise restriction enzyme sites. One such modified MopEH* sequence contains a BspHI site at the stop codon of MopEH*, another one a NdeI site internally in MopEH*, causing some minor changes in the MopEH* sequence. The DNA sequences of these two specific modified MopEH* sequences are fiven as Seq. ID no. 16 and 17, respectively. Diagrams thereof are shown in FIG. 13. Three MopEH*-nodavirus capsid constructs were made, as shown in FIG. 14.

DNA from Atlantic halibut Nodavirus was a gift from Audun Nerland. Based on the published capsid sequence (Accession number AJ245641, Seq. ID no 18 in the attatched patentIn file) the primers listed in table 6 were ordered from Sigma-Aldrich.

TABLE 6 List of primers used to create the Atlantic halibut Nodavirus capsid and MopEH* fusion proteins Primer Sequence Comment AHNVC-F_NcoI GCAAACCATGGTAAGAAATTG Contains a NcoI restriction enzyme site GCTAAACCAGCGACCAC AHNVC-R_NcoI TTAGTCCATGGAGTCAGCTCG Contains a NcoI restriction enzyme site GGTGTTGAG AHNVC-mopE-mut1 GCCATGGgAGTCAGCTCGGGT Inserts a C-residue to correct a phase shift GTTGAG in the mopEH* sequence AHNVCC-F_BspHI TCATGATACATTCGCTCCAAT Contains a BspHI restriction enzyme site CCTAAC AHNVC-R_BspHI TTAGTCTCATGAGTCAGCTCG Contains a BspHI restriction enzyme site GGTGTTGAG AHNVCC_BspHI a CTCCAAGCCTACATTCGCTCC Corrects a frame shift in the mopEH*-AHNVCc mut fusion sequence AHNV 20 aa peptide CTAGCTCATTAGATCGGCCGC Forward strand of a syntetic 20 amino acid fwd TGTCCATTGACT fragment of AHNVC ACAGTCTGGGCACTGGTGATG TCGACCGTGCCG AHNV 20 aa petide CTAGCGGCACGGTCGACATCA Reverse strand of a syntetic 20 amino acid rev CCAGTGCCCAG fragment of AHNVC ACTGTAGTCAATGGACAGCGG CCGATCTAATGAG AHNVCC NheI t GCGTGGCTAGCACATTCGCTC Corrects a frame shift in the mopEH*-AHNVCc mut1 C fusion sequence AHNVCC NheI t CCGAGCTGACGCTAGCGAGCT Corrects a frame shift in the mopEH*-AHNVCc mut2 C fusion sequence

Also two new versions of the mopEH* expressions system where made. One version with a BspHI restriction site replacing the stop codon of mopEH* (Seq. ID no 16) and a second version where a NheI restriction site where mutated into a predicted surface loop of MopEH* (Seq. ID no. 17). The mopEH* plasmids are base don the pBBR1-MCS2 plasmid, and Seq. ID no 19 shows the original, unmodefied pBBR1-mopEH*.

In the first construct, AHNVC-MopEH*, the known sequence of Atlantic halibut Nodavirus capsid protein (AHNVC) were fused to MopEH* using the NcoI restriction enzyme site at the start of MopEH* giving a protein of 72.4 kDa. The leader sequence should be cleaved off when exported from the cytoplasm giving a protein of 72.5 kDa. The DNA and protein sequences of AHNVC-MopEH* are given as Seq. ID 10 and 11, respectively.

In the second construct, MopEH*-AHNVCc, the predicted surface part of Atlantic halibut Nodavirus capsid protein (AHNVCc) were fused to MopEH* using the BspHI restriction enzyme site at the end of MopEH* giving a protein of 52.5 kDa. The leader sequence should be cleaved off when exported from the cytoplasm giving a protein of 49.5 kDa. The DNA and protein sequences of MopE*-AHNVCc are given as Seq. ID no 12 and 13, respectively.

In the third construct, MopEH*-AHNVC-20, aa a 20 amino acid fragment of Atlantic halibut Nodavirus capsid protein (AHNVC-20aa) were inserted into a predicted surface loop of MopEH* using the NdeI restriction enzyme site giving a protein of 41.5 kDa. The leader sequence should be cleaved off when exported from the cytoplasm giving a protein of 38.5 kDa. The DNA and protein sequences of MopEH*-AHNVC-20aa are given as Seq. ID no 14 and 15, respectively.

Expression of Atlantic Halibut Nodavirus Capsid MopEH* Recombinant Proteins

The tree recombinant proteins where conjugated into M. capsulatus ΔmopEH*. To express the recombinant protein M. capsulatus ΔmopEH were grown in a low copper medium. At late log phase growth the cultures were harvest and fractionated into:

1: Spent medium (S) 2: Periplasmic fraction (P) 3: Cytoplasmic fraction (C) 4: Inner membrane (I) 5: Outer membrane (O)

FIG. 15 shows the scheme for localization of the recombinant MopEH*.

The presence of recombinant protein where checked with protein-immunoblot using either AHNV antibodies or MopEH antibodies. The results are given in FIG. 16. As can bee seen in FIG. 16 a, for the MopEH*-AHNVCc construct the majority of recombinant MopEH*-AHNVcc was degraded to MopEH*, although some intact fusion protein is left. FIG. 16 b shows that for the MopEH*-AHNVC 20 aa peptid the MopEH*-AHNVC seems to be misfolded. FIG. 16 c shows that the AHNVC-MopEH* seems to be intact, although the plasmid is a bit unstable.

FIG. 17 shows the results of using antibodies against the MopEH*-AHNVC 20 aa peptid. This clearly shows that the translocated MopEH*-AHNVC is antigenic.

In conclusion, the above results thus show that MopEH* fusion proteins can be successfully constructed, and successfully translocated through the outer membrane of M. capsulatus and there expressed. 

1. A recombinant vector comprising a first nucleotide sequence Seq. ID no. 4, or sequences homologous thereto, excluding sequences comprising the N-terminal sequence present in Seq. ID no 3 but not in Seq. ID no
 4. 2. The recombinant vector according to claim 1, wherein the nucleotide sequence further comprises a second nucleotide sequence.
 3. The recombinant vector according to claim 2, wherein said second nucleotide sequence has multiple cloning sites, said multiple cloning sites being positioned such that insertion of a third nucleotide sequence into said cloning site operable links said third nucleotide sequence to said first nucleotide sequence.
 4. The recombinant vector according to claim 3, wherein said third nucleotide sequence codes for a desired protein or peptide.
 5. The recombinant vector according to claim 4, wherein said protein or peptide is a drug, an antigen or an antibody.
 6. The recombinant vector according to claim 1, wherein said nucleotide sequence further comprises a gene encoding a selection marker.
 7. The recombinant according to claim 6, wherein said selection marker is an antibiotic selection marker.
 8. The recombinant vector according to claim 1, wherein said nucleic acid further comprises a replication origin that function in the host M. capsulatus.
 9. The recombinant vector according to claim 8, wherein said replication origin is smmo or pmmo.
 10. The recombinant vector according to claim 1, wherein the desired protein is expressed in the host M. capsulatus.
 11. The recombinant vector according to claim 10, wherein the desired protein is expressed on the surface of the outer membrane of M. capsulatus.
 12. The recombinant vector according to claim 11, wherein the nucleotide coding for the desired protein contains a region which codes for a peptide stretch functioning as a substrate for a hydrolyzing enzyme capable of cleaving the desired protein from the remaining of the membrane anchored protein, such that the desired protein is excreted to the culture medium.
 13. The recombinant vector according to claim 1, wherein the recombinant vector is a plasmid.
 14. The recombinant vector according to claim 13, wherein the plasmid is pBBmopEH*, with Seq. ID no. 9 or sequences homologous thereto.
 15. The recombinant vector according to claim 1, where sequences homologous to the Seq. ID's have a sequence similarity at least 80%, preferably 85%, more preferably 90% and most preferably 95%.
 16. The recombinant vector according to claim 1, comprising a sequence chosen from the group consisting of Seq. ID no. 10, 12 and
 14. 17. A bacterial host cell transformed with the recombinant vector according to claim
 1. 18. The bacterial host cell according to claim 17, wherein the bacterial cell is M. capsulatus.
 19. A method for producing a desired protein in a bacterial host cell, said method comprising transforming a bacterial host cell with a recombinant vector comprising a first nucleotide sequence Seq. ID no. 4 or sequences homologous thereto, excluding sequences comprising the N-terminal sequence present in Seq. ID no 3 but not in Seq. ID no 4, and said vector further comprising a nucleotide sequence encoding said protein, said further nucleotide sequence being operably linked in frame to said first nucleotide sequence, and culturing said transformed host cell in a suitable medium under conditions allowing expression of said protein.
 20. The method according to claim 19, wherein the method further comprises the step of recovering the expressed protein or peptide from the medium.
 21. The method according to claim 19, wherein the host cell is M. capsulatus.
 22. The method according to claim 19, wherein the desired expressed protein is a drug, wherein said drug is extracted from the host cell, or used together with the host cell for the manufacturing of a vaccine, wherein said vaccine optionally is for oral administration.
 23. The method according to claim 19, wherein sequences homologous to the Seq. ID's are at least 80%, preferably 85%, more preferably 90% and most preferably 95% identical.
 24. A protein capable of being exposed on the surface of a methanotrophic bacterium, wherein the protein is encoded for by Seq. ID no. 4 or sequences homologous thereto, excluding sequences comprising the N-terminal sequence present in Seq. ID no 3 but not in Seq. ID no
 4. 25. The protein according to claim 24, wherein the bacterium is M. capsulatus.
 26. A fusion protein comprising a protein or peptide sequence encoded for by a nucleotide sequence Seq. ID no. 4 or sequences homologous thereto, excluding sequences comprising the N-terminal sequence present in Seq. ID no 3 but not in Seq. ID no 4, and a desired protein or peptide, capable of being translocated.
 27. The fusion protein according to claim 26, where the amino acid sequence of the protein is Seq. ID no 11, 13 or
 15. 28. A protein according to any of claim 24, wherein sequences homologous to the Seq. ID's have a sequence similarity of at least 80%, preferably 85%, more preferably 90% and most preferably 95%. 